Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping

High-spatial-resolution in situ mapping of biomolecules within tissue reveals critical insights into the complex molecular landscape and spatial organization of biological systems. Mass spectrometry imaging (MSI) is a powerful tool for spatially resolved molecular analysis of biological samples, with ongoing demand for improved spatial resolution. Tissue expansion combined with MSI (TEMI) is a recently developed approach that enables multiomics molecular mapping across various biological tissues with significantly improved spatial resolution. Unlike conventional methods that depend on instrument-based enhancements in spatial resolution, TEMI physically enlarges tissue samples via harsh-condition-free hydrogel expansion, achieving more than 3.5-fold increase in effective imaging resolution using standard MSI instrumentation. TEMI delivers single-cell spatial resolution in tissue samples and enables detection of biomolecular heterogeneity that remains uncharacterizable in unexpanded tissue using conventional MSI. Notably, TEMI supports high-spatial-resolution mapping of multiple biomolecular classes—including lipids, metabolites, N-glycans, peptides and proteins—within a single tissue sample. Here, we provide a detailed, step-by-step guide for TEMI, including hydrogel-based tissue expansion under mild conditions, cryosectioning of the expanded tissue-hydrogel sample, a comprehensive experimental workflow for multiomics TEMI on a single tissue section, data acquisition and visualization pipelines, as well as troubleshooting tips. Overall, we demonstrate that TEMI overcomes the long-standing spatial limitations of MSI without requiring hardware modifications, ensuring compatibility with existing MSI instruments and promoting broad accessibility and adoption within the research community. Tissue expansion mass spectrometry imaging enables the mapping of lipids, metabolites, proteins, peptides and N-glycans at high spatial resolution.